Acute Myeloid Leukemia Cells Cells Research Articles

Vacuolar ATPase Is a Possible Therapeutic Target in Acute Myeloid Leukemia: Focus on Patient Heterogeneity and Treatment Toxicity.

Vacuolar ATPase (V-ATPase) is regarded as a possible target in cancer treatment. It is expressed in primary acute myeloid leukemia cells (AML), but the expression varies between patients and is highest for patients with a favorable prognosis after intensive chemotherapy. We therefore investigated the functional effects of two V-ATPase inhibitors (bafilomycin A1, concanamycin A) for primary AML cells derived from 80 consecutive patients. The V-ATPase inhibitors showed dose-dependent antiproliferative and proapoptotic effects that varied considerably between patients. A proteomic comparison of primary AML cells showing weak versus strong antiproliferative effects of V-ATPase inhibition showed a differential expression of proteins involved in intracellular transport/cytoskeleton functions, and an equivalent phosphoproteomic comparison showed a differential expression of proteins that regulate RNA processing/function together with increased activity of casein kinase 2. Patients with secondary AML, i.e., a heterogeneous subset with generally adverse prognosis and previous cytotoxic therapy, myeloproliferative neoplasia or myelodysplastic syndrome, were characterized by a strong antiproliferative effect of V-ATPase inhibition and also by a specific mRNA expression profile of V-ATPase interactome proteins. Furthermore, the V-ATPase inhibition altered the constitutive extracellular release of several soluble mediators (e.g., chemokines, interleukins, proteases, protease inhibitors), and increased mediator levels in the presence of AML-supporting bone marrow mesenchymal stem cells was then observed, especially for patients with secondary AML. Finally, animal studies suggested that the V-ATPase inhibitor bafilomycin had limited toxicity, even when combined with cytarabine. To conclude, V-ATPase inhibition has antileukemic effects in AML, but this effect varies between patients.

Treatment with the tellurium compound AS101 inhibits acute myeloid leukemia cells (AML) invasion and migration

Adhesion molecules, such as integrins, enable acute myeloid leukemia (AML) cells, which travel in the bloodstream, to adhere to the vessels walls. This adhesion is a prerequisite for extra vascular leukemic cell infiltration. The penetrating cells reach vital organs, proliferate and damage the functionality of the tissue which aggravates the patient's condition. The Tellurium compound, AS101, is known for its ability to inhibit specific integrins by Oxidizing key cysteines within the integrins. In this study we found that AS101 can reduce the attachment of human U937 AML cell that expresses high levels of the a4b1 (VLA‐4) integrin to its ligands FN and VCAM‐1. furthermore, AS101 also inhibited the attachment of U937 to the HUVEC human endothelial cells, which express high levels of VCAM‐1 in the presents of TNF‐α. Exposure of U937 cells to AS101 inhibited the pFAK‐ pAKT‐ pGSK signaling, known to be involved in the penetration and migration of cells. AS101 is shown to inhibits the secretion of active MMP‐2 and MMP‐9, enabling cells to penetrate the ECM. Importantly, AS101 could prevent infiltration and migration of AML cells, provided they highly express VLA‐4. This was demonstrated by using patients' AML cells that express high or low levels of the VLA‐4 integrin. In vivo, AS101 prevents invasion of AML cells to the spleen and increase the survival of mice significantly when combined with the chemotherapy ara‐c. This result suggests that the AS101 compound may delay and even prevent the infiltration of the leukemic cells and by doing so, to increase the chances for recovery of the AML patients.

The anti-cancer effect of flaxseed lignan derivatives on different acute myeloid leukemia cancer cells

Flaxseeds have been known for their anti-cancerous effects due to the high abundance of lignans released upon ingestion. The most abundant lignan, secoisolariciresinol diglucoside (SDG), is ingested during the dietary intake of flax, and is then metabolized in the gut into two mammalian lignan derivatives, Enterodiol (END) and Enterolactone (ENL). These lignans were previously reported to possess anti-tumor effects against breast, colon, and lung cancer.This study aims to investigate the potential anti-cancerous effect of the flaxseed lignans SDG, END and ENL on acute myeloid leukemia cells (AML) in vitro and to decipher the underlying molecular mechanism. AML cell lines, (KG-1 and Monomac-1) and a normal lymphoblastic cell line were cultured and treated with the purified lignans. ENL was found to be the most promising lignan, as it exhibits a significant selective dose- and time-dependent cytotoxic effect in both AML cell lines, contrary to normal cells. The cytotoxic effects observed were attributed to apoptosis induction, as revealed by an increase in Annexin V staining of AML cells with increasing ENL concentrations. The increase in the percentage of cells in the pre-G phase, in addition to cell death ELISA analysis, validated cellular and DNA fragmentation respectively. Analysis of protein expression using western blots confirmed the activation of the intrinsic apoptotic pathway upon ENL treatment. This was also accompanied by an increase in ROS production intracellularly. In conclusion, this study demonstrates that ENL has promising anti-cancer effects in AML cell lines in vitro, by promoting DNA fragmentation and the intrinsic apoptotic pathway, highlighting the protective health benefits of flax seeds in leukemia.

JMJD3-regulated expression of IL-6 is involved in the proliferation and chemosensitivity of acute myeloid leukemia cells.

Emerging evidence shows that histone modification and its related regulators are involved in the progression and chemoresistance of multiple tumors including acute myeloid leukemia cells (AML). Our present study found that the expression of histone lysine demethylase Jumonji domain containing-3 (JMJD3) was increased in AML cells as compared with that in human primary bone marrow (HPBM) cells. Knockdown of JMJD3 can decrease the proliferation of AML cells and increase the chemosensitivity of daunorubicin (DNR) and cytarabine (Ara-C). By screening the expression of cytokines involved in AML progression, we found that knockdown of JMJD3 can inhibit the expression of interleukin-6 (IL-6). Recombinant IL-6 (rIL-6) can attenuate si-JMJD3-suppressed proliferation of AML cells. Mechanistically, JMJD3 can positively regulate the promoter activity and transcription of IL-6 mRNA, while had no effect on its mRNA stability. Further, JMJD3 can regulate the expression of p65, which can directly bind with promoter of IL-6 to increase its transcription. Over expression of p65 significantly attenuated si-JMJD3-suppressed expression of IL-6. Collectively, we revealed that JMJD3 can regulate the proliferation and chemosensitivity of AML cells via upregulation of IL-6. It suggested that JMJD3 might be a potential therapy target for AML treatment.

Resistance of leukemia cells to cytarabine chemotherapy is mediated by bone marrow stroma, involves cell-surface equilibrative nucleoside transporter-1 removal and correlates with patient outcome.

The interaction between acute myeloid leukemia cells (AML) with the bone marrow stroma cells (BMSCs) determines a protective environment that favors tumor development and resistance to conventional chemotherapy. We showed that BMSCs secrete soluble factors that protect AML cells from Ara-C induced cytotoxicity. This leukemia chemoresistance is associated with a decrease in the equilibrative nucleoside transporter (ENT1) activity by inducing removal of ENT1 from the cell surface. Reduction of cell proliferation was also observed with activation of AKT and mTOR-dependent cell survival pathways, which may also contribute to the tumor chemoprotection. Analysis of primary BMSC cultures has demonstrated that AML patients with stroma capable to confer Ara-C resistance in vitro compared to AML patients without this stroma capacity were associated with a worse prognosis. The two year overall survival rate was 0% versus 80% respectively (p=0.0001). This is the first report of a chemoprotection mechanism based on the removal of a drug transporter from the cell surface and most importantly the first time that a stroma phenotype has correlated with prognostic outcome in cancer.

The expression of P-gp in leukemia cells is associated with cross-resistance to protein N-glycosylation inhibitor tunicamycin.

In P-gp-positive cell variants obtained from L1210 cells either by selection with vincristine (L1210/R) or by transfection with the human gene encoding P-gp (L1210/T), we have previously described cross-resistance to tunicamycin (TNM), a protein N-glycosylation inhibitor. Here we studied whether this cross-resistance also underlies P-gp-positive variants of human acute myeloid leukemia cells (AML) derived from SKM-1 and MOLM-13 cells (SKM-1/VCR, SKM-1/LEN, MOLM-13/VCR) by selection with vincristine (VCR) and lenalidomide (LEN). While SKM-1/LEN cells were P-gp positive, no P-gp was detected in MOLM-13/LEN cells. P-gp-positive cells could be repeatedly passaged in medium containing TNM. In contrast, more than 90% of P-gp-negative cells were entering and progressing through cell death mechanisms after the third passage in medium containing TNM. Combined apoptosis/necrosis cell death was detected in L1210 cells after exposure to TNM. Passaging of P-gp-negative AML cells in medium containing TNM induced preferentially apoptosis. Damage to P-gp-negative cells induced with TNM was associated with arrest in the G1 phase of the cell cycle. P-gp-positive leukemia cells differed from P-gp-negative cells in the composition of plasma membrane glycoproteins, which we monitored with the aid of different lectins. The application of TNM to cells induced additional changes in membrane-linked glycosides.

RETRACTED: Effect of ST3GAL 4 and FUT 7 on sialyl Lewis X synthesis and multidrug resistance in human acute myeloid leukemia

Sialyl Lewis X (sLe X, CD15s) is a key antigen produced on tumor cell surfaces during multidrug resistance (MDR) development. The present study investigated the effect of α1, 3 fucosyltransferase VII (FucT VII) and α2, 3 sialyltransferase IV (ST3Gal IV) on sLe X oligosaccharides synthesis as well as their impact on MDR development in acute myeloid leukemia cells (AML). FUT7 and ST3GAL4 were overexpressed in three AML MDR cells and bone marrow mononuclear cells (BMMC) of AML patients with MDR by real-time polymerase chain reaction (PCR). A close association was found between the expression levels of FUT7 and ST3GAL4 and the amount of sLe X oligosaccharides, as well as the phenotypic variation of MDR of HL60 and HL60/ADR cells both in vitro and in vivo. Manipulation of these two genes' expression modulated the activity of phosphoinositide-3 kinase (PI3K)/Akt signaling pathway, thereby regulating the proportionally mutative expression of P-glycoprotein (P-gp) and multidrug resistance related protein 1 (MRP1), both of which are known to be involved in MDR. Blocking the PI3K/Akt pathway by its specific inhibitor LY294002 or Akt short hairpin RNA (shRNA) resulted in the reduced MDR of HL60/ADR cells. This study indicated that sLe X involved in the development of MDR of AML cells probably through FUT7 and ST3GAL4 regulating the activity of PI3K/Akt signaling pathway and the expression of P-gp and MRP1.

Ultrasound-Activated Microbubble Cancer Therapy: Ceramide Production Leading to Enhanced Radiation Effect in vitro

Blood vessels within tumours represent a key component for cancer cell survival. Disruption of these vessels can be achieved by inducing vascular endothelial-cell apoptosis. Moreover, endothelial cell apoptosis has been proven to be enhanced by ceramide-increasing drugs. Herein, we introduce a novel therapeutic approach which uses ultrasound-stimulated microbubbles used in combination with radiation to cause a rapid accumulation of ceramide in endothelial cells in-vitro. We also test this modality directly with other cell types as a general method of killing cancer cells. Human umbilical vein endothelial cells (HUVEC), acute myeloid leukemia cells (AML), murine fibrosarcoma cells (KHT-C), prostate cancer cells (PC3), breast cancer cells (MDA-MB-231) and astrocytes were used to evaluate this mechanism of inducing cell death. Survival was measured by clonogenic assays, and ceramide content was detected using immunohistochemistry. Exposure of cell types to ultrasound-stimulated bubbles alone resulted in increases in ceramide for all cell types and survivals of 12 ± 2%, 65 ± 5%, 83 ± 2%, 58 ± 4%, 58 ± 3%, 18 ± 7% for HUVEC, AML, PC3, MDA, KHT-C and astrocyte cells, respectively. Results from selected cell types involving radiation treatments indicated additive treatment enhancements and increases in intracellular ceramide content one hour after exposure to ultrasound-activated microbubbles and radiation. Endothelial cell survival decreased from 8 ± 1% after 2 Gy of radiation treatment alone and from 12 ± 2% after ultrasound and microbubbles alone, to 1 ± 1% with combined treatment. In Asmase +/+ astrocytes, survival decreased from 56 ± 2% after 2 Gy radiation alone and from 17 ± 7% after ultrasound and microbubbles alone, to 5 ± 2% when combined. Using ASMase deficient astrocytes (Asmase -/-) and Sphingosine-1-phosphate (S1P), we also demonstrate that ultrasound-activated microbubbles stimulate ASMase activity and ceramide production. These findings suggest that ultrasound-stimulated microbubbles could be used as a new biomechanical method to enhance the effects of radiation.

Abstract 3501: Flavopiridol decreases Mcl-1 and XIAP in acute myeloid leukemia cell resulting in augmentation of sorafenib-induced apoptosis

Abstract The PI3K/AKT/mTOR and Raf/MEK/ERK pathways, resulting in increased levels of Mcl-1, XIAP and survivin, have been found to be activated in acute myeloid leukemia cells (AML) due to the mutant receptor tyrosine kinases. FMS-like tyrosine kinase (FLT3) mutations including FLT3-internal tandem duplication (FLT3-ITD) are found in about 30% of cases of acute myeloid leukemia and confer an increased relapse rate and reduced overall survival. Sorafenib, inhibiting Raf-1 of the MAPK pathway resulting in diminished MEK and ERK phosphorylation, is being tested in AML patients with FLT3-ITD and modest responses have been observed: similarly flavopiridol, a CDK and mRNA transcription inhibitor, has been tested in AML patients with some evidence of activity. We hypothesize that sorafenib plus flavopiridol will have enhanced therapeutic effects. The combined apoptotic effects of sorafenib plus flavopiridol were investigated in HL-60, MV4;11 FLT3-ITD+ cells, as well as in primary leukemic cells isolated from AML patients. Sorafenib and flavopiridol indepedently induced apoptosis at 10 μM and 200 nM, respectively, in HL-60 cells which was correlated with the down-regulation of Mcl-1, XIAP and/or survivin. The combination of sorafenib (5 μM) plus flavopiridol (100 nM) produced augmented apoptosis associated with enhanced diminution in levels of Mcl-1. MV4;11 cells were more sensitive than HL-60 cells to sorafenib-induced apoptosis, but not to flavopiridol, with the down-regulation of Mcl-1 and survivin. Addition of flavopiridol to MV4;11 cells enhanced sorafenib-induced apoptosis with augmented down-regulation of XIAP. Primary cells isolated from two AML patients with FLT3-ITD+ cells exhibited augmented apoptosis induction with the combination treatment of sorafenib plus flavopiridol. Our data suggest that the augmented apoptosis induction in AML cells by sorafenib plus flavopiridol is mediated by decreasing levels of complimentary anti-apoptotic proteins. This novel combination therapy is worthy of a clinical trial in FLT3-ITD+ AML patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3501. doi:10.1158/1538-7445.AM2011-3501

In-Vitro Culture of Human CD80/IL2 Lentivirus Transduced Acute Myeloid Leukemia Cells (AML) Promote NK Cell Activation and Cytolytic Activity

Natural killer (NK) cells are increasingly recognized as an important component in the graft versus leukemia response following allogeneic hematopoietic stem cell transplantation. Immunotherapeutic strategies aim to promote NK cell activity however, the presence of regulatory T cells (Tregs) which inhibit effector immune responses pose a potential challenge to the efficacy of such regimens. We have previously shown that ‘in-vitro’ culture of AML cells transduced with a self-inactivating lentivirus (LV) encoding CD80 (B7.1) and IL2 enhance allogeneic (allo) and autologous (auto) T cell proliferation and cytotoxicity. The effect on NK cell activity and Tregs has not previously been studied and is of particular importance as IL2 stimulates NK cell and Treg activity. Peripheral blood mononuclear cells (PBMCs) from healthy donors (allo) or AML patients (auto) were cultured for 7 days ‘in-vitro’ with either unmodified or LV-CD80/IL2 AMLs. The number of NK cells (CD3−CD56+) and Tregs (CD3+CD4+CD25highFoxp3+) was examined by multi-color flow cytometry. We observe an increase in the number of NK cells (p<0.001) with an increase in the expression of the activation receptors NKp30, NKp44, CD244, CD25, CD69 and HLA-DR following allo culture with LV-CD80/IL2 AML compared with unmodified AML. Autologous culture provides a weaker stimulus ‘in-vitro’ however, a higher number of NK cells (p=0.002) and a consistent increased expression of the activation receptors NKp30, NKp44, NKp46, NKG2D, NKG2C and CD69, as well as up regulation of the cytolytic marker CD107a was detected following auto stimulation with LV-CD80/IL2 AML compared with unmodified AML. Up regulation of CD107a was also observed in allo cultures stimulated with both unmodified and LV-CD80/IL2 AML cells. In contrast, a consistent increase in the number of Tregs was observed following allo (p=0.043) but not auto (p=0.515) LV-CD80/IL2 AML culture. Foxp3 may be unregulated on activated CD4+ T cells therefore the number of CD3+CD4+CD25highFoxp3+CD27+ Tregs was also examined. An increase in the number of CD27+ Tregs was observed following allo (p=0.017) but not auto (p=0.807) LV-CD80/IL2 AML cell culture. A standard 51Cr release assay was used to examine cytotoxicity against primary unmodified AMLs on days 0 and 7 following LV-CD80/IL2 AML cell culture. Tregs are capable of suppressing CD4+ and CD8+ T cell and NK cell cytotoxicity, therefore lysis of unmodified AMLs was initially examined using whole PBMCs as effectors. Even in the presence of Tregs an increase in lysis of allo unmodified AMLs was observed: 2.2% day 0, 4.6% following culture with unmodified AMLs; 20.4% following LV-CD80/IL2 AML cell culture. Importantly, an increase in lysis of auto AML was also detected: 0% day 0, 2.1% unmodified AML culture, 16% LV-CD80/IL2 AML culture. The ratio of Tregs to effector T cells is important for the suppressive function of Tregs. The number of Tregs in the cytotoxicity assays is likely to be lower than that required for a significant suppressive effect to be observed. We next examined the cytotoxicity of NK cells using K562 and unmodified AMLs as targets. NK cells were negatively isolated on days 0 and 7 following either unmodified AML or LV-CD80/IL2 AML cell culture and used as effectors in a 51Cr release assay. In keeping with the changes in NK cell activation receptor expression, we demonstrate a significant increase in NK cell cytotoxicity against both K562 and primary unmodified AMLs. Lysis of K562 increased from 46.7% on day 0 to 90.4% after LV-CD80/IL2 stimulation. Importantly, an increase in lysis of both allo and auto unmodified AMLs was detected following LV-CD80/IL2 AML cell culture. Lysis of allo AMLs increased from a median of 11.8% on day 0, 8.7% following culture with unmodified AML to 20.1% following LV-CD80/IL2 AML cell culture using a low effector: target ratio of just 5:1. Importantly, an increase in lysis of auto AML from 0.4% on day 0, 2.1% with unmodified AML cells to 21.5% following LV-CD80/IL2 AML stimulation was observed. LV-CD80/IL2 AML cells enhance NK cell activation and cytotoxicity against allo and auto unmodified AMLs. Furthermore, cytotoxicity is enhanced even in the presence of Tregs with an increase in Tregs only observed following allo culture. Vaccination of patients with LV-CD80/IL2 AML cells therefore represents a potential strategy to promote T and NK cell cytotoxicity and enhance anti-leukemia immune responses in patients with AML.

Effects of human bone marrow stromal cell line (HFCL) on the proliferation, differentiation and apoptosis of acute myeloid leukemia cell lines U937, HL-60 and HL-60/VCR

This study investigated the effects of human bone marrow fibroblastoid stromal cell line (HFCL) on the proliferation, differentiation and chemosensitivity of acute myeloid leukemia cells (AML) in vitro coculture. By setting up coculture system of sensitive U937, HL-60 cell line and multidrug-resistant (MDR) HL-60/VCR cells in direct contact with human bone marrow fibroblastoid stromal cell line HFCL, or separated by transwell, the proliferation of AML cells cocultured with HFCL cells was inhibited, compared with AML cells alone. And NBT positive cells increased slightly. The percentage of G1 phase cells of AML cells cocultured with HFCL cells was higher than that without HFCL cells, and that of S phase cells was lower. The expression of CD11b and CD14 increased. Meanwhile HL-60 and HL-60/VCR cells treated by TPT were observed to have apoptosis characteristic morphological changes. The proportion of G0/G1 HL-60 and HL-60/VCR cells treated with TPT increased and the sub-G1 increased. The percentage of Annexin V-positive cells and apoptotic cells increased with expression of activated Caspase-3 and the reduced expression of Bcl-2. But when they were cocultured with HFCL cells, the percentage of Annexin V-positive cells and apoptotic cells decreased and sub-G1 reduced. After indirect contact with HFCL cells the expression of activated Caspase-3 decreased and the expression of Bcl-2 increased. After direct contact with HFCL cells for 96 h, the expression levels of 582 genes in HL-60 cells were up-regulated, and 1,323 genes were down-regulated at least twofold by Affymetrix GeneChip Human Genome U133 set A. The expression change in some genes, such as HL14, was confirmed by RT-PCR and northern blot. In a word, HFCL cells could inhibit the proliferation, induce the monocytic differentiation of U937, HL-60 and HL-60/VCR cells, and prevent TPT-induced apoptosis in HL-60 and HL-60/VCR cells via modulation of Bcl-2 and active Caspase-3. Many genes might take part in the influence of HFCL cells on AML cells, which may give important insights into the interaction of bone marrow stromal cells and leukemic cells.

Autophagy and Apoptosis Are Induced Simultaneously in Leukemia Cells by Vitamin K2.

Autophagy is a membrane trafficking process that leads to degradation of cytosolic proteins and organelles by lysosomes, and it is conserved throughout evolution. Autophagy is induced above basal levels in response to nutrient deprivation or trophic factor withdrawal, and it sustains metabolism through the targeted catabolism of long-lived proteins. Thus, autophagy acts as a self-limited survival mechanism. However, recent findings have suggested that autophagy is also as a potential contributor to programmed cell death.We previously reported that vitamin K2 (VK2) selectively and potently induces the apoptosis of cultured acute myeloid leukemia cells (AML) and AML cell lines, but has almost no effect on normal hematopoietic cells. In addition, the clinical benefit of using VK2 for patients with AML and myelodysplastic syndrome has been reported. In the present study, we focused on whether autophagy was induced in AML cells by treatment with VK2.HL-60bcl-2 cells with stable transfection of the bcl-2 gene showed 5-fold greater expression of BCL-2 compared with HL-60neo cells, and these cells were resistant to apoptosis induced by VK2, along with the occurrence of monocytic differentiation via G1 arrest. This suggested that VK2 had a differentiation-inducing effect on AML cells that were resistant to apoptosis. However, a cytocidal effect was still detectable in a caspase-independent manner. Treatment of HL-60bcl-2 cells with VK2 resulted in an increase of acid vesicular organelles (AVO) detected by acridine orange staining (for lysosomes).Immunonoblotting with an antibody for microtubule-associated protein-1 light chain-3 (LC3) revealed the conversion of LC3-I into LC3-II a form that migrates more rapidly on SDS-PAGE and participates in the formation of autophagosomes (one of the hallmarks of autophagy). Electron microscopy revealed an increase of autophagosome formation after exposure to VK2. Finally, the induction of caspase-independent cell death by VK2 was suppressed in the presence of 3-methyladenine. These results suggest that autophagic death of HL-60bcl-2 cells was induced by VK2. Furthermore, detailed electron microscopic assessment of HL-60neo cells during exposure to VK2 revealed that autophagosome formation was more prominent compared with that occurring in H-60bcl-2 cells, although nuclear chromatin condensation was also observed at the same time. These findings indicated a mixture of the morphologic features of apoptosis and autophagy. The present results suggest that autophagy and apoptosis can both be induced simultaneously by VK2. However, autophagy only became detectable when cells were protected from rapid apoptotic death.

Fucose Binding Lectin for Characterizing Acute Myeloid Leukemia Progenitor Cells

The reactivity of acute myeloid leukemia cells (AML) was determined in 29 patients using the fucose binding lectin Ulex europaeus agglutinin (UEA) as surface marker. We show a marked heterogeneity in the UEA-binding abilities of the cells in these patients as determined by fluorescence analysis of the blasts labeled with the UEA coupled to the fluorescent molecule FITC. The results suggest a correlation between the capability of AML blast cells to bind UEA and cytologic maturation, because in 1 of 10 M1, 3 of 8 M2, 6 of 8 M4, and 1 of 3 M5 cytology types UEA binding to the leukemic cells was apparent. In 13 cases, the cells gave rise to colonies in vitro. The amount of UEA binding to AML colony-forming cells (AML-CFU) was determined by cell sorting and subsequent colony culture of UEA-negative, intermediately positive, and highly fluorescent cells. AML-CFU from none of the four patients with M1 cytology were UEA positive, whereas they showed intense reactivity with the lectin in 1 of 4 cases with M2 cytology and in all 4 cases of M4. In these five cases with strongly UEA positive AML-CFU, the fluorescence distribution of the colony formers differed from that of the total leukemia population, indicating that AML-CFU represent a subpopulation of AML cells with specific UEA-binding properties. Normal bone marrow myeloid and multipotential colony-forming cells (CFU-GM, CFU-GEMM) showed low or no binding of UEA. UEA-FITC appears a useful reagent for membrane analysis of AML-CFU. In certain cases, UEA-FITC labeling may be applied to discriminate AML-CFU from normal hematopoietic progenitors.

Fucose binding lectin for characterizing acute myeloid leukemia progenitor cells

The reactivity of acute myeloid leukemia cells (AML) was determined in 29 patients using the fucose binding lectin Ulex europaeus agglutinin (UEA) as surface marker. We show a marked heterogeneity in the UEA- binding abilities of the cells in these patients as determined by fluorescence analysis of the blasts labeled with the UEA coupled to the fluorescent molecule FITC. The results suggest a correlation between the capability of AML blast cells to bind UEA and cytologic maturation, because in 1 of 10 M1, 3 of 8 M2, 6 of 8 M4, and 1 of 3 M5 cytology types UEA binding to the leukemic cells was apparent. In 13 cases, the cells gave rise to colonies in vitro. The amount of UEA binding to AML colony-forming cells (AML-CFU) was determined by cell sorting and subsequent colony culture of UEA-negative, intermediately positive, and highly fluorescent cells. AML-CFU from none of the four patients with M1 cytology were UEA positive, whereas they showed intense reactivity with the lectin in 1 of 4 cases with M2 cytology and in all 4 cases of M4. In these five cases with strongly UEA positive AML-CFU, the fluorescence distribution of the colony formers differed from that of the total leukemia population, indicating that AML-CFU represent a subpopulation of AML cells with specific UEA-binding properties. Normal bone marrow myeloid and multipotential colony-forming cells (CFU-GM, CFU-GEMM) showed low or no binding of UEA. UEA-FITC appears a useful reagent for membrane analysis of AML-CFU. In certain cases, UEA-FITC labeling may be applied to discriminate AML-CFU from normal hematopoietic progenitors.